The Trypanosoma brucei RNA-Binding Protein TbRRM1 is Involved in the Transcription of a Subset of RNA Pol II-Dependent Genes.

It has been long thought that RNA Polymerase (Pol) II transcriptional regulation does not operate in trypanosomes. However, recent reports have suggested that these organisms could regulate RNA Pol II transcription by epigenetic mechanisms. In this paper, we investigated the role of TbRRM1 in transcriptional regulation of RNA Pol II-dependent genes by focusing both in genes located in a particular polycistronic transcription unit (PTU) and in the monocistronic units of the SL-RNA genes. We showed that TbRRM1 is recruited throughout the PTU, with a higher presence on genes than intergenic regions. However, its depletion leads both to the decrease of nascent RNA and to chromatin compaction only of regions located distal to the main transcription start site. These findings suggest that TbRRM1 facilitates the RNA Pol II transcriptional elongation step by collaborating to maintain an open chromatin state in particular regions of the genome. Interestingly, the SL-RNA genes do not recruit TbRRM1 and, after TbRRM1 knockdown, nascent SL-RNAs accumulate while the chromatin state of these regions remains unchanged. Although it was previously suggested that TbRRM1 could regulate RNA Pol II-driven genes, we provide here the first experimental evidence which involves TbRRM1 to transcriptional regulation.

TbRRM1 knockdown produces abnormal cell morphology and apoptotic-like death in the bloodstream form of T. brucei.

TbRRM1, an SR-related protein, is involved in transcriptional and post-transcriptional gene expression regulation in procyclic T. brucei. In previous work, we found that TbRRM1 is essential and its depletion leads to cell cycle impairment, aberrant phenotypes and cell loss by apoptotic-like death. Here, we report the findings obtained after TbRRM1 knockdown in bloodstream parasites. Depletion of TbRRM1 in this cell stage led also to growth arrest and cell loss by apoptosis-like death. However, microscopic analysis showed aberrant cell morphology with parasites displaying flagellum detachment and cytokinesis impairment after RNAi induction, suggesting that TbRRM1 could play different roles depending on parasite stage.

The protein family TcTASV-C is a novel Trypanosoma cruzi virulence factor secreted in extracellular vesicles by trypomastigotes and highly expressed in bloodstream forms.

TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes.

Depletion of the SR-Related Protein TbRRM1 Leads to Cell Cycle Arrest and Apoptosis-Like Death in Trypanosoma brucei.

Arginine-Serine (RS) domain-containing proteins are RNA binding proteins with multiple functions in RNA metabolism. In mammalian cells this group of proteins is also implicated in regulation and coordination of cell cycle and apoptosis. In trypanosomes, an early branching group within the eukaryotic lineage, this group of proteins is represented by 3 members, two of them are SR proteins and have been recently shown to be involved in rRNA processing as well as in pre-mRNA splicing and stability. Here we report our findings on the 3rd member, the SR-related protein TbRRM1. In the present study, we showed that TbRRM1 ablation by RNA-interference in T. brucei procyclic cells leads to cell-cycle block, abnormal cell elongation compatible with the nozzle phenotype and cell death by an apoptosis-like mechanism. Our results expand the role of the trypanosomal RS-domain containing proteins in key cellular processes such as cell cycle and apoptosis-like death, roles also carried out by the mammalian SR proteins, and thus suggesting a conserved function in this phylogenetically conserved protein family.

Antibodies against the Trypanosoma cruzi ribosomal P proteins induce apoptosis in HL-1 cardiac cells.

High levels of antibodies (Abs) against the C-terminal end of the Trypanosoma cruzi ribosomal P2ß protein, defined by the R13 peptide, are detected in sera from patients with chronic Chagas heart disease (cChHD). These Abs can cross-react with the ß1-adrenergic receptor (ß1-AR), inducing a functional response in cardiomyocytes. In this study, we report that a monoclonal Ab against the R13 peptide, called mAb 17.2, and its single-chain Fv fragment (scFv), C5, caused apoptosis of murine adult cardiac HL-1 cells, and this effect was inhibited by pre-incubation with the ß-blocker, propranolol. In addition, apoptosis induced by mAb 17.2 might involve the mitochondrial pathway evidenced by an increase in pro-apoptotic molecule, Bax/anti-apoptotic molecule, Bcl(XL), mRNA levels. HL-1 cells also underwent apoptosis after incubation with nine of 23 IgGs from cChHD patients (39.1%) that presented reactivity against R13 peptide and ß1-AR. The apoptotic effect caused by these IgGs was partially abolished by pre-incubation with R13 peptide or propranolol, suggesting the involvement of the C-terminal end of ribosomal P proteins and the ß-adrenergic pathway. Moreover, we observed high rates of cardiomyocyte apoptosis in two tissue samples from cChHD patients by using a TUNEL assay and staining of active caspase-3. Our data demonstrate that Abs developed during T. cruzi infection have a strong cardiomyocyte apoptosis inducing ability, which could contribute to the heart disease developed in patients with cChHD.